Abstract Background Organizational Health Literacy (OHL) implies responsiveness and friendliness of healthcare organizations. In the context of the WHO Action Network M-POHL, Italy evaluated OHL in a hospital as a pilot. Methods The international self-assessment tool OHL-Hos explores 8 standards using 141 items. It was firstly translated and culturally adapted to Italy, and then tested to assess OHL in the public general hospital of Pistoia (Central Italy). A physician, 6 staff members of the healthcare management unit, and 3 medical residents in public health from the Florence University were involved in the pilot. The Pistoia hospital has 1,300 employees (44% nurses, 24% physicians) to face 18,000 hospitalizations per year. Results Completing the questionnaire took more than 5 hours. A comprehensive agreement was reached, even though different approaches aiming at improving HL in healthcare and non-healthcare staff emerged. Scores resulted acceptable/good for standard 5 (communication with patients) and 7 (promoting staff HL with regard to occupational risks and personal lifestyles), low for standards 1 (implementing OHL overall the hospital), 2 (involving stakeholders in developing documents, materials, services), 3 (staff training for personal and organizational HL) and 8 (contributing to local population personal HL improvement), which would apply more peculiar for other settings than hospitals. Conclusions The participation of the hospital working group in the assessment using the OHL-Hos raised interest toward this matter. It results time consuming for its excessive length, and some standards do not fit to the Italian hospital context, resulting more useful to assess the territorial organization. Reduction of the number of items and revision/reformulation of some of them are suggested. Nonetheless, the tool proved to be useful in indicating the most critical areas needing ad hoc interventions, such as training all the hospital staff.